Cryptococcus neoformans is an important conditioned pathogen, and often infects patients with low immunocompetence or immunodeficiency, causing deep fungal infection with a principle symptom of central nervous system infection. The deep fungal infection may cause an extremely high case fatality rate. Large series of epidemiologic study performed by Center for Disease Control and Prevention (CDC) in 1992-1993 showed that an annual incidence rate of the deep fungal infection is 178.3/million, wherein the annual incidence rate of cryptococcosis is 65.5/million, accounting for about 36.7%.
In recent years, because of long-term wide applications of broad-spectrum antibacterial drugs, adrenocortical hormone, tumor chemotherapy, radiation oncology and immunosuppressors after organ transplantation as well as the epidemic of AIDS, the cryptococcosis is obviously increased. Cryptococcus neoformans meningitis (hereinafter referred to as cryptococcal meningoencephalitis) is the most common central nervous system disease caused by the Cryptococcus neoformans, and accounts for about 80% of the cryptococcosis. Due to complicated clinical manifestations and atypical symptoms, the cryptococcal meningoencephalitis is difficult to be diagnosed. About 80% of patients suffering from the cryptococcal meningoencephalitis may be misdiagnosed with tubercular meningitis.
Primary causal factors of the Cryptococcus neoformans are capsule, melanin, growth capability thereof in a host temperature environment, etc. The capsule is one of the main determination factors that influence toxicity of the Cryptococcus neoformans. A main component of the capsule is capsular polysaccharide and has the effects of inhibiting phagocytosis and activating complement, and the component with the highest content is glucuronoxylomannan (GXM). In an early diagnosis method of the cryptococcus neoformans meningitis, Wang et al. detect a Cryptococcus neoformans capsular polysaccharide antigen by adopting a latex agglutination test and evaluate diagnostic sensitivity and specificity by fungal culture and direct microscopic examination methods. Results show that the sensitivities of the latex agglutination test, the fungal culture and the direct microscopic examination are respectively 91.1%, 69.6% and 73.2%, and the specificities are respectively 96.0%, 100% and 100%. Thus, it is believed that, the latex agglutination test for detecting the Cryptococcus neoformans capsular polysaccharide antigen can serve as the early diagnosis method of the cryptococcus neoformans meningitis. The method for performing cryptococcus latex agglutination test on the cryptococcus neoformans meningitis has high specificity in diagnosis of the cryptococcus neoformans meningitis, so that an early diagnosis rate is increased (Wang H, Yuan X, Zhang L. Latex agglutination: Diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen. Pakistan journal of pharmaceutical sciences, 2015, 28(1 Suppl): 307-311). Therefore, the Cryptococcus neoformans capsular polysaccharide is very important to early diagnosis of the cryptococcus neoformans meningitis.
Kozel et al. perform the following steps: culturing the cryptococcus neoformans in a culture tank, performing autoclaved sterilization on a culture solution, centrifuging to remove thalli, filtering the supernatant with a filter membrane of 0.45 um, performing ultrafiltration concentration, adding sodium acetate and glacial acetic acid into the concentrated solution, and precipitating a coarse capsular polysaccharide with ethanol; repeatedly extracting with chloroform/n-butyl alcohol (v:v=5:1), removing protein, precipitating the capsular polysaccharide with ethanol, centrifuging and collecting the precipitate, dissolving the precipitate in deionized water, dialyzing and freeze-drying, thereby obtaining refined capsular polysaccharide. (Kozel T R, Cazin J. Nonencapsulated variant of Cryptococcus neoformans I. Virulence studies and characterization of soluble polysaccharide. Infection and immunity, 1971, 3(2): 287-294). Lots of chloroform should be used in the above method. The chloroform is a toxic and harmful dangerous chemical, has carcinogenic risk and also has high requirements for operating conditions, protection of operators, sewage treatment and the like.
Bryan et al. perform the following steps: washing cryptococcus neoformans cells with distilled water for 3 times, centrifuging and collecting the cells, resuspending the collected wet cells with 15 mL of dimethyl sulfoxide (DMSO) and incubating for 30 minutes; precipitating and separating the cells by centrifuging, dialyzing DMSO supernatant for 12 hours, exchanging water every 2 hours, fully dialyzing the obtained sample with distilled water or 1 mM EDTA for 3 days, and performing freeze drying on a polysaccharide solution obtained by dialyzing, thereby obtaining the capsular polysaccharide (Bryan R A, Zaragoza O, Zhang T, et al. Radiological studies reveal radial differences in the architecture of the polysaccharide capsule of Cryptococcus neoformans. Eukaryotic Cell, 2005, 4(2): 465-475). The above method is long in preparation cycle, and the yield of the product is low.
Maxson et al. perform the following steps: centrifuging the culture fluid of the cryptococcus cells, separating a cell precipitate, concentrating the obtained supernatant by about 20 times by using a polyether sulfone ultrafiltration plate, continuously stirring in the concentrating process, discharging a fluid phase after a sticky film is formed on the filtration plate, recovering binding material by using a cell scraper, and finally performing freeze drying, thereby obtaining the capsular polysaccharide (Maxson M E, Dadachova E, Casadevall A, et al. Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule. Eukaryotic cell, 2007, 6(1): 95-109). The above method is simple in operation and short in preparation cycle, but the yield of the product is also low.
In addition, Frases et al. find in experiments that, extracellular polysaccharide and the capsular polysaccharide of the cryptococcus are structurally different, and also find in experiments that, by comparing polysaccharide prepared by precipitating through cetyl trimethyl ammonium bromide with polysaccharide prepared by concentration and ultrafiltration (see an extraction method in Maxson M E, Dadachova E, Casadevall A, et al. Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule. Eukaryotic cell, 2007, 6(1): 95-109), the mass is greater, and conformations of the polysaccharides are different. Then, it is judged that, a common method for extracting the Cryptococcus neoformans capsular polysaccharide may obviously influence the structure and antigenicity of the product (Frases S, Nimrichter L, Viana N B, et al. Cryptococcus neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical, chemical, and antigenic differences. Eukaryotic cell, 2008, 7(2): 319-327).
At present, there is an urgent need to develop a preparation method of the Cryptococcus neoformans capsular polysaccharide with convenience, high efficiency, low cost and high specificity in the field, and the preparation method shall effectively avoid the use of any toxic and harmful chemical reagent. The present invention overcomes the defects in the prior art, provides a preparation method of Cryptococcus neoformans capsular polysaccharide GXM, the prepared GXM, and a detection kit prepared by utilizing the GXM. The prepared GXM has high purity and specificity. The detection kit prepared by utilizing the antigen has high detection sensitivity.